Composition comprising antimicrobial metal ions and a quaternary cationic surfactant

ABSTRACT

The present invention relates to an antimicrobial composition suitable for use on skin and wounds comprising a source of an antimicrobial metal ion and a quaternary cationic surfactant.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.15/620,639, filed Jun. 12, 2017, filed on Jun. 12, 2017, which is acontinuation of Ser. No. 14/114,517, filed on Nov. 14, 2013, now issuedas U.S. Pat. No. 9,675,077 on Jun. 13, 2017, which is a U.S. NationalPhase of PCT/GB12/00329, filed Apr. 5, 2012, which claims the benefit ofGB1105829.4, filed on Apr. 6, 2011, each of which is entirelyincorporated herein by reference.

This invention relates to an antimicrobial composition which can beapplied to skin, wounds, cuts, abrasions or burns for the prevention ortreatment of infections or to wound dressings and the like forapplication to skin, wounds, cuts, abrasions or burns. More particularlythe invention relates to a composition capable of providing effectiveantimicrobial activity while at the same time avoiding wound and skinirritation and retardation of wound healing.

Overuse of antibiotics and the associated increase in bacterialresistance is impacting the efficacy of antibiotics in the treatment ofwound infection. Effective alternatives to antibiotics are thusdesirable.

Topical antimicrobial materials and preparations containing them havelong been recognised as playing an important part in minimising theopportunity for skin and wound infections. Non-antibiotic antimicrobialsare non-selective chemical agents that can be safe to use on livingtissue. Molecular iodine, ionic silver and oxidising agents such assodium hypochlorite and chlorine dioxide have been recognised asantimicrobial agents with effectiveness against a wide range ofmicro-organisms. There are however several barriers to making aneffective antimicrobial composition for application to wounds based onsuch agents. One problem is that these antimicrobial agents tend toreact with organic materials found in the wound other than the intendedmicrobial targets. This means that to be effective, antimicrobial agentsneed to be included in treatment compositions at high levels, which maycause undesirable side effects with prolonged use such as cell toxicity,hypersensitivity reactions, skin staining and systemic effects. Suchside effects are further described in “In vitro cytotoxity of silver:implication for clinical wound care”. Poon V K, Burd A. Burns. 2004March; 30(2):140-7, “A review of iodine toxicity reports”. Pennington IA. J Am Diet Assoc. 1990 November; 90(11):1571-81 and “Topicalantimicrobial toxicity”. Lineaweaver W, Howard R, Soucy D, McMorris S,Freeman J, Crain C, Robertson J, Rumley T. Arch Surg. 1985 March;120(3):267-70.

There is therefore a need for a means to make treatment compositionseffective without simply increasing the level of antimicrobial agentincluded in the composition. It has also been recognised that woundbacteria often exist in biofilms and that these are more difficult totreat than their planktonic counterparts.

We have found that it is possible to increase the effectiveness ofantimicrobial metal ions by including a quaternary cationic surfactantin the formulation.

Accordingly a first aspect of the invention provides an antimicrobialcomposition suitable for use on skin and wounds comprising a source ofantimicrobial metal ion and a quaternary cationic surfactant.

The presence of the quaternary cationic surfactant enhances the effectof the antimicrobial metal ion so that the performance of theantimicrobial metal ion is improved. For instance the presence of thequaternary cationic surfactant can increase the speed at which theantimicrobial metal ion works

By the term antimicrobial it is meant a substance that inhibits thegrowth of, or kills, micro-organisms from the taxonomical kingdoms ofbacteria, fungi and protozoa. An effective antimicrobial composition istherefore one which is used to reduce and prevent the spread andproliferation of micro-organisms in a specific application. In woundcare this can be interpreted in terms of control of cross-infection,prevention or elimination of infection and the reduction of recalcitrantbioburden that can cause delayed healing and chronicity.

We have also found that it is possible to prepare a composition whichincludes a quaternary cationic surfactant which is effective under theconditions of pH normally found in a wound.

Accordingly a second aspect of the invention provides a compositionsuitable for use on wounds comprising a quaternary cationic surfactantat a pH of 4 to 8.

The compositions according to a first aspect of the invention comprisean antimicrobial agent, preferably a metal ion for example silver, iron,nickel, copper, chromium, manganese, gold, gallium, magnesium, mercury,lead, aluminium, lead, zinc, bismuth, tin and palladium. Preferably themetal ion is silver. The antimicrobial agent is preferably included inthe composition at a level of from 0.01% to 10% by weight, morepreferably 0.1% to 5% and even more preferably 0.5% to 1.5% by weight or1% to 5%. If the composition is in aqueous solution the antimicrobialmetal ion is preferably in an aqueous solution comprising from 0.00001%to 1.0% by weight or more preferably 0.0001% to 0.1%, even morepreferably 0.0001% to 0.02% by weight or 0.001% to 1.0% by weight.

The compositions according to the invention comprise a cationicsurfactant. The cationic surfactant can be a quaternary ammonium salt,an alkyl pyridinium salt, an alkyl imidazolium salt, an alkylmorpholinium salt, a benzethonium salt or an ethoxylated quaternaryammonium salt or mixtures thereof. Preferably where the salt is aquaternary ammonium salt, it is selected from the group of monoalkyltrimethyl ammonium salts, dialkyl dimethyl ammonium salts and monoalkylmonobenzyl dimethyl ammonium salts. Preferably the cationic surfactantis a quaternary cationic surfactant and more preferably a quaternaryammonium surfactant. Preferably the cationic surfactant is selected fromthe group of benzethonium, benzalkonium, dimethyldialkylonium,alkylpyridinium and alkyltrimethylammonium cations with any counter ion,for example: bromide, chloride, acetate or methyl sulphate. Preferablythe quaternary cationic surfactant is present at a level of more than orequal to 0.025% by weight, more preferably from 0.05% to 4% by weightand more preferably from 0.5% to 2% by weight.

The pH of the composition is preferably between 4 and 8, more preferablybetween 4 and 6 and most preferably between 4.5 and 5.5. The desired pHmay be achieved by incorporating buffering agents in the composition.Examples of buffering agents which may be included are citricacid/di-sodium hydrogen phosphate, citric acid/sodium citrate, aceticacid/sodium acetate. The buffering agent may conveniently be present inan amount of about 0.5% to 2% by weight of the composition so as toprovide an isotonic composition.

The composition preferably comprises ethylenediaminetetra-acetic acid(EDTA). EDTA is preferably present as the di-, tri- or tetra-basic saltsof EDTA. We have found that these salts enhance the antimicrobial effectof the ionic metal in disrupting biofilm. For example we have found thatEDTA at concentrations of 0.25-0.5% weight by volume was effective inmaking a range of microorganisms in the biofilm state vulnerable toantimicrobial agents.

EDTA is preferably present in the compositions of the present inventionat a level of 0.1% to 4% by weight of the composition, more preferablyless than 2% by weight, more preferably 0.2 to 1% by weight.

The compositions of the present invention may be in the form of asolution which can be used as a spray to be applied to dressingmaterials or a solution dip into which dressing materials can beimmersed, or in the form of thin soluble films which can be laminated towound dressings or used along with a separate dressing in the form of akit. Alternatively the compositions can be in the form of a softsemi-solid such as a gel, foam or creme which maintains a moist woundhealing environment and promotes natural healing. A soft semi-solidformulation gives the advantage of being able to flow into a wound toform an intimate contact with the wound bed and provide antimicrobialeffects to the entire surface of a wound. Preferably the formulation hasa high enough viscosity that it does not flow out of a wound onto othertissues. Preferably the pH of the formulation is buffered at around 5.5as this does not alter the pH balance of the peri-wound tissue andtherefore protects it. The compositions of the invention may also bepresent as a dry powder spray.

The following examples are illustrative of the present invention.

The data relevant to the examples is represented in the accompanyingdrawings which show:

FIGS. 1a, 1b, 1c and 1d show grey scales representing antimicrobialefficacies of a range of silver concentrations against a range ofsurfactant concentrations and a range of surfactants and optionalingredients:

FIG. 2 shows a comparison of the depth of penetration (mm) ofantimicrobial action between silver dressings tested both with treatmentand without treatment of a composition according to the invention; and

FIG. 3 shows a comparison of the anti biofilm activity (MBEC data) ofsolutions and semi-solid gels.

EXAMPLE 1

Quaternary Cationic Surfactant Enhancement of Silver Efficacy

Method: The MBEC Assay System using the Calgary Biofilm Device providesan assay for screening antibiotics and biocides for activity againstbacterial biofilms. The system involves a reactor for the formation of96 equivalent biofilms. The MBEC Assay System is suited to determinationof MBEC values (Minimum Biofilm Eradication Concentration) and otherrelated values. A description of the system and method is given in “TheMBEC Assay System: Multiple Equivalent Biofilms for Antibiotic andBiocide Susceptibility Testing” by Howard Ceri, Merle Olson, DouglasMorck, Douglas Storey, Ronald Read,

Andre Buret and Barbara Olson, 2001 Methods in Enzymology Vol 337, [25]p 377 and “The Calgary Biofilm Device: New Technology for RapidDetermination of Antibiotic Susceptibilities of Bacterial Biofilms”Ceri, Olson, Stremick, Read, Morck and Buret Journal of ClinicalMicrobiology, June 1999, Vol 37, No. 6, p 1771-1776.

Biofilms were grown on the pegs of 96-well plate lids (Nunc-TSP parts445497 and 167008, Thermo Fisher Scientific Inc); these were thenthoroughly rinsed in purified water. Into a fresh plate, using aseptictechniques and filter sterilised solutions, aliquots of an aqueoussilver nitrate solution, various quaternary cationic surfactant aqueoussolutions and purified water were pipetted to give a matrix of 10411test samples of various silver and surfactant concentrations. Thebiofilm covered plate lids were then replaced and left in contact withthe test solution for either 30 minutes, 2 or 4 hours. After this timethe lids were removed and any residual test agent was removed from thepegs by rinsing in sterile normal saline solution (0.85% wlw NaCl). Theresidual biofilms were then physically removed from the pegs by theestablished sonication method. The released and still viable bacteriawhere then grown-on in their planktonic form for 24 hours in a new platein which the wells contained a growth medium. The concentration ofsurviving bacteria released from the pegs was then estimated byturbidity measurement on an optical plate reader. Although opticaldensities were recorded, a simple grey scale representation was adequatefor interpretation.

Tested silver ion concentrations ranged from 1.625 μg/ml to 200 μg/ml,doubling in concentration between successive samples. The cationicsurfactants tested were benzethonium benzalkonium chloride,dimethyldialkylammonium chloride, cetyltrimethylammonium bromide andcetylpyridinium chloride at concentrations ranging from 31.25 μg/ml to200011 g/till also doubling in concentration between successive samples.

Results: The results are presented by the charts in FIG. 1a and by thelefthand diagram in FIGS. 1b, 1c and 1d . The grey scale represents theantimicrobial efficacy. The higher the efficacy the lighter the shade ofgrey so that no antimicrobial efficacy is represented by black and highantimicrobial efficacy is represented by light grey. Positive synergiesare represented by a shift to a paler position on the grey scale fromthe experimental control (no surfactant) presented in FIG. 1a . In thefigures silver concentration is shown on the y-axis and surfactantconcentration is shown on the x-axis.

These results show the concentration ranges where synergy was observed.All cationic surfactants assayed showed synergy with ionic silver abovea specific critical concentration for each surfactant. Below thiscritical concentration inhibition of the antimicrobial effect of silverions was observed.

EXAMPLE 2

EDTA and Quaternary Cationic Surfactant Enhancement of Silver Efficacy

Method: As Example 1 but with each test solution also containing 0.25%w/w di-sodium ethyl enediaminetetra-acetic acid salt.

Results: The results are presented in FIGS. 1b, 1c and 1d by the diagramon the righthand side. Diagrams on the lefthand side are thecorresponding experiments without EDTA.

These results show that EDTA further enhances the synergisticantimicrobial activity of the cationic surfactant and the metal ion.

EXAMPLE 3

Enhancement of Antimicrobial Efficacy of Examples of Silver ContainingWound Dressings

The depth to which an antimicrobial effect on an agar gel containingStaphylococcus aureus was assessed for different types of treated anduntreated silver containing wound dressings.

Materials:

Test Dressings:

-   -   AQUACEL Ag (5×5 cm), batch 9L019035, absorbent, gelling,        fibrous-felt dressing containing 1.2% w/w ionic silver.    -   Allevyn Ag Non-adhesive, batch 0935, absorbent foam dressing        containing silver sulphadiazine.    -   Silvercel Hydro-alginate, batch, 37923, a dressing comprised of        a mixed alginate and silver-coated nylon fibrous pad wrapped in        a perforated ethylene methyl acrylate film.    -   Sorbsan Silver—Plus, batch 012035, an absorbent nonwoven        alginate pad impregnated with silver, bonded to a secondary        absorbent viscose layer.    -   All of the above treated with di-sodium EDTA and benzethonium        chloride.    -   Microbiological Media:        -   Maximal Recovery Diluent (MRD)        -   Pre-dried Tryptone Soy Agar (TSA) plates        -   0.85% Saline Solution        -   Molten Tryptone Soy Agar (TSA)        -   Industrial Denatured Alcohol (IDA)        -   Sterile Deionised Water (SDW)    -   Microbial Challenge: Staphylococcus aureus NCIMB 9518

Methods: Two 3.75 cm diameter circular samples were aseptically cut fromeach dressing. One of each sample was placed into a sterile Petri dishfor later testing. The remaining sample was treated with a solutionaccording to the invention using the following procedure: —0.25 gramseach of di-sodium EDTA and benzethonium chloride were weighed intoseparate sterile bottles and dissolved in 100 ml of 50:50 (v/v) IDA:SDWand 100% IDA respectively. 50 μg/cm² of EDTA and 50 fig/cm² ofbenzethonium chloride were added to the dressing by carefully pipetting220 μl of each solution over the entire surface of each dressing sample.The samples were then placed back into the original packaging and driedin a vacuum oven at 90° C. and 0.9 atmospheres of vacuum forapproximately 2 hours.

A colorimeter was used to prepare a suspension of Staphylococcus aureusapproximately 1×10⁸ CFU/ml in MRD (0.16-0.18 OD540) and serially dilutedto obtain approximately 1×10⁴ CFU/ml. Two 100 ml volumes of molten TSA(approximately 45° C.) were each inoculated with 1 ml of the 1×10⁴CFU/ml Staphylococcus aureus suspension and swirled to mix. 20 mlvolumes of this bacterially seeded molten agar were then measured andpoured into nine 60 ml pots (with an internal diameter of 3.75 cm). Whencooled and set these pots were incubated at 35° C.±3° C. for 4 hours±15minutes to initiate growth. After 4 hours each of the test dressing washydrated with 2.5 ml of 0.85% Saline Solution in a sterile Petri dishand placed into a 60 ml pot and onto the surface of the seeded agar. Onepot had no dressing applied to act as a positive control. The pots werethen incubated for another 24 hours after which time the dressings wereremoved and disposed of. The pots were then re-incubated for a minimum72 hours to allow the already established colonies to grow. After thefinal incubation period the pots were evaluated and photographed next toa calibrated rule.

Results: Where the dressing had imparted an antibacterial effect in theseeded agar beneath the dressing the agar appeared transparent. Wherebacterial growth had not been inhibited the agar appeared opaque. Thedepth of the transparent zone of agar from the surface in contact withthe dressing was interpreted as an indication of antimicrobial efficacy.Results are presented in FIG. 2. The positive control was opaque to thesurface of the agar, i.e. there was no transparent zone. The resultsindicate that all of the silver containing dressings tested had someantimicrobial potency, but this varied between dressing types.

The addition of this example formulation of the invention increased thedepth of the transparent zone by at least a factor of two for alldressing types but the rank order remained the same. This suggested thatpotency is dependent on dressing type but synergystic enhancement wasindependent of dressing type.

The Aquacel Ag had a greater depth of penetration than the otherdressings for both the control and treated tests. Aquacel Ag also hadthe greatest improvement in antimicrobial penetration when treated witha solution according to the invention as the average depth ofpenetration was three times greater than the control.

EXAMPLE 4

Enhancement of Antimicrobial Efficacy with Different AntimicrobialMetals

Previous Examples using the MBEC (Minimum Biofilm EradicationConcentration) method have shown that the addition of specificantimicrobial agents to silver enhances its antimicrobial activity. Thepurpose of this example was to assess the effect of these agents on theantimicrobial activity of other metals, using the MBEC method.

Method: A 0.2% w/w aqueous stock solution of the quaternary cationicsurfactant didecyl dimethyl ammonium chloride (DDAC) was prepared from aconcentrated commercial solution (50% w/v solution, Merck KGaA,Darmstadt, Germany). Individual stock solutions of silver nitrate,copper (II) nitrate trihydrate, gallium (III) nitrate, nickel (II)nitrate hexahydrate, zinc sulphate heptahydrate, manganese (II) chloridetetrahydrate, iron (II) sulphate heptahydrate, iron (III) sulphatehydrate, and copper (II) nitrate trihydrate were prepared fromcommercially available laboratory solids. Each solution was adjusted toapproximately pH 5.5 by the addition of small amounts of dilute aqueoussodium hydroxide and/or dilute hydrochloric acid. The metal ionsolutions were then volumetrically diluted to 0.1% w/w (with respect tothe metal) with purified water. Any precipitates were kept homogenouslysuspended by vigorous shaking. The MBEC assay as described in Example 1was then performed for the surfactant solution alone, each individualmetal ion solution and then for each metal solution in combination withthe surfactant solution.

Results:

MBEC for the metal ion (ppm) t Without With 0.1% Metal pH DDAC DDACSynergy Silver 5.27   12.5   0.097 +ve Copper * 5.47   15.6    1.95 +veIron (II) * 5.30 >1000  ≤0.24 +ve Iron (III) * 5.40 >1000  ≤0.24 +veGallium * 5.51 >1000  ≤0.24 +ve Manganese 5.40 >1000  ≤0.24 +ve Nickel5.51 >1000  ≤0.24 +ve Zinc 5.40 >1000   ≤125 +ve DDAC —   0.2% -na- -na-† Lowest concentration at which activity was observed; ppm is equivalentto μg/g (or μg/ml in aqueous solutions). These values are approximate. *These metals formed precipitates when the 0.1% solutions were pHadjusted to pH 5.5; however the precipitate was dissolved when dilutedin the test plate.

Literature data for metal solutions (pH unadjusted) suggests thefollowing order in terms of antimicrobial activity:

-   -   Silver>Iron>Nickel>Copper>Gallium>Magnesium>Bismuth

The MBEC for DDAC alone was determined as approximately 0.2%. When 0.1%DDAC was used in combination with the listed metal ion solutions allproduced a significant reduction in the MBEC for the metal thusdemonstrating a synergistic effect which is independent of the identityof antimicrobial metal used.

EXAMPLE 5

The Effect of pH

When used at high concentration many cationic surfactants haveantimicrobial activity; typically this is enhanced by an alkaline pH.Similarly, many researchers have found that the activity of some silvercompounds is also enhanced by elevated pH. To prevent pain and tissuedamage, products applied to broken skin need to be near pH neutral orhave slightly acidic in pH. This Example investigates the effect of pHon the synergystic effect between a quaternary cationic surfactant andan antimicrobial metal.

Method: The following aqueous stock solutions were prepared:—benzethonium chloride (1.0% w/w), silver nitrate (0.1% w/w with respectto the metal), sodium acetate (0.5M) and acetic acid (0.5M).

In the control experiment sodium acetate and acetic acid solutions weremixed at various ratios and diluted with purified water to give a rangeof pH buffer solutions with different pH's but with the same overallionic strength (0.1M with respect to acetate ion). To each, sufficientsilver nitrate solution was added to make the solution 0.01% w/w withrespect to silver. Each solution was then challenged in the MBEC methodas described in Example 1 to determine if, at this level of pH andsilver, the solution was above or below the MBEC.

Using a second set of similarly prepared solutions (0.1M acetate buffer,0.01% silver) a second MBEC experiment was performed in which theconcentration of the surfactant solution was varied.

Results: None of the 0.01% silver solutions pH buffered in the range 4.7to 7.7 were effective against biofilms in the MBEC model. Surfactantconcentrations of >0.25%, 5_ 0.10% and 0.025% were required to eradicatebiofilm in the MBEC model at pH 6.9, 6.2 and 5.5 respectively.

This suggests the synergystic antimicrobial effect of the currentinvention is enhanced by reducing pH (becoming more acidic) which iscounter to current wisdom.

EXAMPLE 6

Enhancement of the Antimicrobial Efficacy of a Semi-Solid (Gel)Composition Containing Silver, EDTA and Quaternary Surfactant

Previous Examples (1, 2, 4 & 5) illustrate applicability to simplesolutions. This example seeks to demonstrate that the addition of inertexcipients that modify the physical form and properties of the baseactive solution have no effect on the observed efficacy. Increasing theviscosity by the addition of the gelling agent hydroxyethylcellulose(HEC, Aqualon type: Natrosol 2501{X Pharm) is used in this example withthe activity of the formulated gels being compared to the equivalentsolution using the MBEC assay as previously described.

Method: Stock solutions of silver nitrate, benzethonium chloride,di-sodium EDTA (pH ˜4), tri-sodium EDTA (pH ˜8) and HEC were prepared.These were then combined in various ways to produce a matrix of samplesof one, two, three components (EDTA being included only once in anycombination) with and without HEC. The final component concentrationsbeing 0%, 0.0001% or 0.02% for silver (Ag); 0%, 0.1% or 1% forbenzethonium chloride (BeCl); 0%, 0.2% or 1% for EDTA; 0% or 0.1% forHEC. Test samples were challenged against a microbial biofilm using theMBEC method previously outlined in Example 1. The results were recordedas either effective (no bacterial growth) or ineffective (bacterialgrowth as indicated by turbidity). All test samples were prepared induplicate and each MBEC determination was performed in triplicatetherefore six assays results were obtained for each sample.

Results:

A summary of the results for combinations are shown in FIG. 3. Adding aninert excipient (HEC) to modify the physical properties of the testsubstance did not alter the anti-biofilm activity of the test mixture.Synergistic behaviour was still observed for the combinations at pH 4and pH 8 at the lower end of the preferred concentration ranges. Noinhibitory effects of the addition of the inert excipient were observedat the upper end of the preferred concentration ranges.

1. An antimicrobial composition for treating wound bacteria in abiofilm, the composition comprising ionic silver, EDTA at a level offrom 0.01% to 10% by weight, and a quaternary cationic surfactant,wherein the quaternary cationic surfactant is present at a level of morethan or equal to 0.025% by weight, wherein the composition has a pH from4 to 8, wherein the ionic silver, the EDTA, and the quaternary cationicsurfactant are present in the composition in amounts that providesynergistic antimicrobial activity against the wound bacteria, andwherein the composition is in the form of a thin soluble film, the thinsoluble film being suitable for lamination to a wound dressing.
 2. Acomposition as claimed in claim 1, wherein the ionic silver is presentat a level of from 0.00001% to 1.0% by weight of the composition.
 3. Acomposition as claimed claim 1, wherein the cationic surfactant isselected from a group of the salts where the cation is benzethonium,benzalkonium, dimethyldiakylonium, alkylpyridinium andalkyltrimethylammonium.
 4. A composition as claimed in claim 1, whereinthe quaternary cationic surfactant is present at a level of from 0.05%to 4% by weight.
 5. A composition as claimed in claim 1, wherein theEDTA is present as a di-, tri- or tetra-basic salt of EDTA.
 6. Acomposition as claimed in claim 1, wherein the EDTA is present in thecomposition at a level of 0.1% to 4% by weight of the composition.
 7. Aprocess for making an antimicrobial wound dressing comprising: (i)obtaining an absorbent wound dressing, and (ii) treating the wounddressing with an antimicrobial composition in the form of a thin solublefilm, the thin soluble film being laminated to the wound dressing andfor treating wound bacteria in a biofilm, wherein the compositioncomprises ionic silver, EDTA at a level of from 0.01% to 10% by weight,and a quaternary cationic surfactant, wherein the quaternary cationicsurfactant is present at a level of more than or equal to 0.025% byweight, wherein the composition has a pH from 4 to 8, and wherein theionic silver, the EDTA, and the quaternary cationic surfactant arepresent in the composition in amounts that provide synergisticantimicrobial activity against the wound bacteria.
 8. A composition asclaimed in claim 1, wherein the quaternary cationic surfactant isselected from benzethonium chloride and benzalkonium chloride.
 9. Acomposition as claimed in claim 1 having a pH from 4.5 to 5.5.
 10. Acomposition as claimed in claim 1, wherein the quaternary cationicsurfactant is present at a level of from 0.5% to 2% by weight.
 11. Acomposition as claimed in claim 1, wherein the EDTA is present in thecomposition at a level of 0.2% to 1% by weight.
 12. A composition asclaimed in claim 1, having a pH of about 5.5 to protect peri-woundtissue by not altering its pH balance.
 13. A process as claimed in claim12, wherein the composition has a pH from 4.5 to 5.5.
 14. A process asclaimed in claim 12, wherein the composition has a pH of about 5.5 toprotect peri-wound tissue by not altering its pH balance.
 15. A processas claimed in claim 12, wherein the EDTA is present in the compositionat a level of 0.2% to 1% by weight.